Protein ID (NP or XP #) or Wolbachia#: ZP_04462052.1
Organism (including strain): Yersenia pestis Etiologic Risk Group (see link below): Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia Disease Information (sort of like the Intro to your Mini Research Write up):
The Bubonic Plague is very well known to many for its disastrous effects on medieval Europe. Yersenia pestis, a bacterium, was identified in 1894 to be the cause of this horrible disease. The plague is still alive today, seen mostly in the Americas, Asia, and Africa. Treatment for the disease is very effective when diagnosed early on. However, drug-resistant strains were identified in 1995, and from then on, there have been more drug-resistant strains emerging. Along with this, many fear it can be used as a biological weapon. So, there has been pressure to find new drug targets rather quickly. http://www.sciencedirect.com/science/article/pii/S0969212611004114
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361726/ Essentiality of this protein: Catalyzes last step of the bacterial fatty acid biosynthesis (FAS-II) pathway Is it a monomer or multimer as biological unit? Monomer Complex of proteins?: Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Isoniazid, one of the front-line drugs against tuberculosis, and triclosan, a broad-spectrum antiseptic, both inhibit the last and rate limiting step of the FAS-II pathway, where enoyl-ACP reductase is necssary. FabV, an isoform of enoyl-ACP reductase, is found in Y. pestis. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361726/ EC#: 1.3.1.9 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=1.3.1.9&Suchword=&organism[]=Yersinia+pestis&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay (see Sigma links below) -- -or link (or citation) to paper that contains assay information http://www.jbc.org/content/270/44/26538.long -- links to assay reagents (substrates) pages. http://openwetware.org/wiki/Image:140302_NAD_NADP_NADH_NADPH_assay_info.jpg --- List cost and quantity of substrate reagents, supplier, and catalog #
NAD/NADH 10 mL Catalog # G9071 $467.00
NAD/NADH 50 mL Catalog # G9072 $1870.00 Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 3ZU5
Current Inhibitors:
1-(2-chlorobenzyl)-4-hexylpyridin-2(1H)-one
1-(3-amino-2-methylbenzyl)-4-hexylpyridin-2(1H)-one
triclosan
Expression Information (has it been expressed in bacterial cells): Expressed in B. pseudomallei Purification Method: Image of protein (PyMol with features delineated and shown separately): Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
length of your protein in Amino Acids: 405 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 43986.8 Molar Extinction coefficient of your protein at 280 nm wavelength: 48610 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
ATGATTATCAAGCCGCGTGTACGTGGTTTTATCTGTGTTACTGCGCATCCAACGGGTTGCGAGGCTAACGTGAA
AAAACAGATCGACTACGTTACCACCGAAGGTCCGATTGCGAACGGTCCGAAACGTGTTCTGGTTATTGGTGCG
TCTACCGGTTACGGTCTGGCAGCCCGTATCACGGCGGCTTTTGGTTGCGGCGCAGATACCCTGGGCGTTTTCTT
CGAACGTCCTGGCGAAGAAGGCAAACCGGGTACGTCTGGTTGGTACAATTCTGCGGCGTTCCACAAATTCGCT
GCGCAAAAAGGTCTGTACGCGAAATCTATCAACGGTGATGCGTTCTCTGACGAAATTAAGCAGCTCACCATTGA
CGCGATCAAACAGGACCTCGGTCAGGTTGACCAAGTTATTTACTCTCTCGCGTCTCCACGTCGTACCCACCCGA
AGACCGGTGAAGTTTTCAACTCTGCGCTGAAACCTATTGGTAATGCTGTAAATCTGCGTGGCCTGGACACCGAC
AAAGAAGTTATCAAAGAATCTGTCCTGCAGCCGGCGACCCAGTCTGAAATCGACTCTACCGTTGCGGTCATGGG
CGGTGAGGATTGGCAAATGTGGATCGATGCCCTCCTGGACGCGGGTGTTCTCGCGGAAGGTGCCCAAACCACC
GCGTTCACCTACCTGGGTGAGAAAATCACGCACGATATCTATTGGAACGGTTCTATCGGTGCGGCGAAAAAAGA
CCTGGACCAGAAAGTTCTGGCGATCCGTGAATCTCTGGCGGCTCACGGTGGCGGCGATGCTCGTGTTTCTGTTC
TGAAAGCGGTTGTTACCCAGGCGTCTTCTGCGATCCCTATGATGCCGCTCTACCTGTCTCTGCTGTTCAAAGTTAT
GAAAGAGAAAGGTACGCATGAAGGTTGCATCGAGCAGGTCTACTCCCTCTACAAAGATTCTCTGTGCGGTGATT
CTCCGCACATGGACCAAGAAGGTCGTCTCCGTGCGGACTACAAGGAGCTCGACCCTGAAGTCCAGAATCAGG
TTCAGCAGCTGTGGGACCAGGTGACCAACGACAACATCTACCAACTGACGGATTTCGTGGGTTACAAATCTGAA
TTCCTCAACCTCTTCGGTTTCGGTATCGACGGTGTTGACTACGACGCGGATGTTAACCCGGACGTCAAAATCCCG
AATCTGATCCAGGGTTAA GC% Content for gene: 52.5% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): ATGATTATCAAGCCGCGTGTACGTGGTTTTATCTGTGTTACTGCGCATCCAACGGGTTGCGAGGCTAACGTGAAAA
AACAGATCGACTACGTTACCACCGAAGGTCCGATTGCGAACGGTCCGAAACGTGTTCTGGTTATTGGTGCGTCTA
CCGGTTACGGTCTGGCAGCCCGTATCACGGCGGCTTTTGGTTGCGGCGCAGATACCCTGGGCGTTTTCTTCGAAC
GTCCTGGCGAAGAAGGCAAACCGGGTACGTCTGGTTGGTACAATTCTGCGGCGTTCCACAAATTCGCTGCGCAA
AAAGGTCTGTACGCGAAATCTATCAACGGTGATGCGTTCTCTGACGAAATTAAGCAGCTCACCATTGACGCGATCA
AACAGGACCTCGGTCAGGTTGACCAAGTTATTTACTCTCTCGCGTCTCCACGTCGTACCCACCCGAAGACCGGTG
AAGTTTTCAACTCTGCGCTGAAACCTATTGGTAATGCTGTAAATCTGCGTGGCCTGGACACCGACAAAGAAGTTAT
CAAAGAATCTGTCCTGCAGCCGGCGACCCAGTCTGAAATCGACTCTACCGTTGCGGTCATGGGCGGTGAGGATTG
GCAAATGTGGATCGATGCCCTCCTGGACGCGGGTGTTCTCGCGGAAGGTGCCCAAACCACCGCGTTCACCTACCT
GGGTGAGAAAATCACGCACGATATCTATTGGAACGGTTCTATCGGTGCGGCGAAAAAAGACCTGGACCAGAAAGT
TCTGGCGATCCGTGAATCTCTGGCGGCTCACGGTGGCGGCGATGCTCGTGTTTCTGTTCTGAAAGCGGTTGTTACC
CAGGCGTCTTCTGCGATCCCTATGATGCCGCTCTACCTGTCTCTGCTGTTCAAAGTTATGAAAGAGAAAGGTACGCA
TGAAGGTTGCATCGAGCAGGTCTACTCCCTCTACAAAGATTCTCTGTGCGGTGATTCTCCGCACATGGACCAAGAA
GGTCGTCTCCGTGCGGACTACAAGGAGCTCGACCCTGAAGTCCAGAATCAGGTTCAGCAGCTGTGGGACCAGGT
GACCAACGACAACATCTACCAACTGACGGATTTCGTGGGTTACAAATCTGAATTCCTCAACCTCTTCGGTTTCGGTAT
CGACGGTGTTGACTACGACGCGGATGTTAACCCGGACGTCAAAATCCCGAATCTGATCCAGGGTTAA *GC% Content for gene (codon optimized): 52.5%
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**
NCBI Gene # or RefSeq#: 372467227
Protein ID (NP or XP #) or Wolbachia#: ZP_04462052.1
Organism (including strain): Yersenia pestis
Etiologic Risk Group (see link below): Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia
Disease Information (sort of like the Intro to your Mini Research Write up):
The Bubonic Plague is very well known to many for its disastrous effects on medieval Europe. Yersenia pestis, a bacterium, was identified in 1894 to be the cause of this horrible disease. The plague is still alive today, seen mostly in the Americas, Asia, and Africa. Treatment for the disease is very effective when diagnosed early on. However, drug-resistant strains were identified in 1995, and from then on, there have been more drug-resistant strains emerging. Along with this, many fear it can be used as a biological weapon. So, there has been pressure to find new drug targets rather quickly.
http://www.sciencedirect.com/science/article/pii/S0969212611004114
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361726/
Essentiality of this protein: Catalyzes last step of the bacterial fatty acid biosynthesis (FAS-II) pathway
Is it a monomer or multimer as biological unit? Monomer
Complex of proteins?:
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
Isoniazid, one of the front-line drugs against tuberculosis, and triclosan, a broad-spectrum antiseptic, both inhibit the last and rate limiting step of the FAS-II pathway, where enoyl-ACP reductase is necssary. FabV, an isoform of enoyl-ACP reductase, is found in Y. pestis.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361726/
EC#: 1.3.1.9
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=1.3.1.9&Suchword=&organism[]=Yersinia+pestis&show_tm=0
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay (see Sigma links below)
-- -or link (or citation) to paper that contains assay information
http://www.jbc.org/content/270/44/26538.long
-- links to assay reagents (substrates) pages.
http://openwetware.org/wiki/Image:140302_NAD_NADP_NADH_NADPH_assay_info.jpg
--- List cost and quantity of substrate reagents, supplier, and catalog #
NAD/NADH 10 mL Catalog # G9071 $467.00
NAD/NADH 50 mL Catalog # G9072 $1870.00
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 3ZU5
Current Inhibitors:
1-(2-chlorobenzyl)-4-hexylpyridin-2(1H)-one
1-(3-amino-2-methylbenzyl)-4-hexylpyridin-2(1H)-one
triclosan
Expression Information (has it been expressed in bacterial cells): Expressed in B. pseudomallei
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
length of your protein in Amino Acids: 405
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 43986.8
Molar Extinction coefficient of your protein at 280 nm wavelength: 48610
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGATTATCAAGCCGCGTGTACGTGGTTTTATCTGTGTTACTGCGCATCCAACGGGTTGCGAGGCTAACGTGAA
AAAACAGATCGACTACGTTACCACCGAAGGTCCGATTGCGAACGGTCCGAAACGTGTTCTGGTTATTGGTGCG
TCTACCGGTTACGGTCTGGCAGCCCGTATCACGGCGGCTTTTGGTTGCGGCGCAGATACCCTGGGCGTTTTCTT
CGAACGTCCTGGCGAAGAAGGCAAACCGGGTACGTCTGGTTGGTACAATTCTGCGGCGTTCCACAAATTCGCT
GCGCAAAAAGGTCTGTACGCGAAATCTATCAACGGTGATGCGTTCTCTGACGAAATTAAGCAGCTCACCATTGA
CGCGATCAAACAGGACCTCGGTCAGGTTGACCAAGTTATTTACTCTCTCGCGTCTCCACGTCGTACCCACCCGA
AGACCGGTGAAGTTTTCAACTCTGCGCTGAAACCTATTGGTAATGCTGTAAATCTGCGTGGCCTGGACACCGAC
AAAGAAGTTATCAAAGAATCTGTCCTGCAGCCGGCGACCCAGTCTGAAATCGACTCTACCGTTGCGGTCATGGG
CGGTGAGGATTGGCAAATGTGGATCGATGCCCTCCTGGACGCGGGTGTTCTCGCGGAAGGTGCCCAAACCACC
GCGTTCACCTACCTGGGTGAGAAAATCACGCACGATATCTATTGGAACGGTTCTATCGGTGCGGCGAAAAAAGA
CCTGGACCAGAAAGTTCTGGCGATCCGTGAATCTCTGGCGGCTCACGGTGGCGGCGATGCTCGTGTTTCTGTTC
TGAAAGCGGTTGTTACCCAGGCGTCTTCTGCGATCCCTATGATGCCGCTCTACCTGTCTCTGCTGTTCAAAGTTAT
GAAAGAGAAAGGTACGCATGAAGGTTGCATCGAGCAGGTCTACTCCCTCTACAAAGATTCTCTGTGCGGTGATT
CTCCGCACATGGACCAAGAAGGTCGTCTCCGTGCGGACTACAAGGAGCTCGACCCTGAAGTCCAGAATCAGG
TTCAGCAGCTGTGGGACCAGGTGACCAACGACAACATCTACCAACTGACGGATTTCGTGGGTTACAAATCTGAA
TTCCTCAACCTCTTCGGTTTCGGTATCGACGGTGTTGACTACGACGCGGATGTTAACCCGGACGTCAAAATCCCG
AATCTGATCCAGGGTTAA
GC% Content for gene: 52.5%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGATTATCAAGCCGCGTGTACGTGGTTTTATCTGTGTTACTGCGCATCCAACGGGTTGCGAGGCTAACGTGAAAA
AACAGATCGACTACGTTACCACCGAAGGTCCGATTGCGAACGGTCCGAAACGTGTTCTGGTTATTGGTGCGTCTA
CCGGTTACGGTCTGGCAGCCCGTATCACGGCGGCTTTTGGTTGCGGCGCAGATACCCTGGGCGTTTTCTTCGAAC
GTCCTGGCGAAGAAGGCAAACCGGGTACGTCTGGTTGGTACAATTCTGCGGCGTTCCACAAATTCGCTGCGCAA
AAAGGTCTGTACGCGAAATCTATCAACGGTGATGCGTTCTCTGACGAAATTAAGCAGCTCACCATTGACGCGATCA
AACAGGACCTCGGTCAGGTTGACCAAGTTATTTACTCTCTCGCGTCTCCACGTCGTACCCACCCGAAGACCGGTG
AAGTTTTCAACTCTGCGCTGAAACCTATTGGTAATGCTGTAAATCTGCGTGGCCTGGACACCGACAAAGAAGTTAT
CAAAGAATCTGTCCTGCAGCCGGCGACCCAGTCTGAAATCGACTCTACCGTTGCGGTCATGGGCGGTGAGGATTG
GCAAATGTGGATCGATGCCCTCCTGGACGCGGGTGTTCTCGCGGAAGGTGCCCAAACCACCGCGTTCACCTACCT
GGGTGAGAAAATCACGCACGATATCTATTGGAACGGTTCTATCGGTGCGGCGAAAAAAGACCTGGACCAGAAAGT
TCTGGCGATCCGTGAATCTCTGGCGGCTCACGGTGGCGGCGATGCTCGTGTTTCTGTTCTGAAAGCGGTTGTTACC
CAGGCGTCTTCTGCGATCCCTATGATGCCGCTCTACCTGTCTCTGCTGTTCAAAGTTATGAAAGAGAAAGGTACGCA
TGAAGGTTGCATCGAGCAGGTCTACTCCCTCTACAAAGATTCTCTGTGCGGTGATTCTCCGCACATGGACCAAGAA
GGTCGTCTCCGTGCGGACTACAAGGAGCTCGACCCTGAAGTCCAGAATCAGGTTCAGCAGCTGTGGGACCAGGT
GACCAACGACAACATCTACCAACTGACGGATTTCGTGGGTTACAAATCTGAATTCCTCAACCTCTTCGGTTTCGGTAT
CGACGGTGTTGACTACGACGCGGATGTTAACCCGGACGTCAAAATCCCGAATCTGATCCAGGGTTAA
*GC% Content for gene (codon optimized): 52.5%
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**